The goal is to develop a technique for labeling antibodies with metallic radionuclides and to determine the relationship between accelerated blood clearance (caused by increased chelating groups per antibody) and the retention of immunological activity. We have compared three methods (DTPA carboxycarbonyl anhydride, cyclic DTPA anhydride and carbodiimide method) of conjugating DTPA to antibody in our attempt to optimize the balance between the number of DTPA groups conjugated to the antibody and the antibody-binding activity. We used a practical concentraton (300 micrograms in 1 ml medium) anti-HSA antibody (Ab). The DTPA carboxycarbonyl anhydride and cyclic DTPA anhydride methods are 2-step synthetic methods using DTPA as a starting compound. The carbodiimide method is, however, a 1-step synthesis using carbodiimide as a coupling agent to conjugate DTPA to amino groups of Ab. DTPA carboxycarbonyl anhydride is very unstable and requires that the reaction be carried out below 4 degrees C. An optimum conjugation giving three indium atoms incorporated per Ab with 65% retention of antibody-binding activity was obtained when an anhydride to antibody ration of 1173 was used. For cyclic DTPA anhydride method, DTPA conjugation was dependent on the pH of the reaction medium. Four buffer solutions were compared: Hepes buffer at pH 7, phosphate buffer at pH 8.6 and bicarbonate buffer at pH 8.2. The bicarbonate buffer was the best in terms of pH stability and indium atoms (11) incorporated per Ab (50% retention of the binding activity occurred). The carbodiimide method was the simplest we have studied and enabled us to incorporate up to 18 indium atoms per Ab with a retention of 40% activity when the reaction was carried out at pH 6.2. The systematic variation of the parameters of these classical reactions using dilute antibody solutions has allowed us to choose the optimal labeling conditions for a particular situation.